PROJECT SUMMARY: Exosomes are secreted extracellular vesicles that contain bioactive molecules such as proteins and microRNAs. The exosomes have recently drawn considerable interest as they are implicated in many pathophysiological processes such as neurodegeneration, viral propagation, and tumor invasion. Despite the great interests in the medical fields, the basic cell biological understanding of the exosomes is disproportionally lacking. The exosomes are generated when the limiting membranes of endosomes invaginate toward the lumen to form multivesicular bodies (MVBs). The Endosomal Sorting Complex Responsible for Transport (ESCRT) mediates the biogenesis of the exosomes. However, how the exosomes are delivered to the plasma membrane for their release remains elusive. The exocyst is an octameric protein complex consisting of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84. The exocyst is thought to mediate the trafficking of protein and lipid cargos from intracellular compartments to the plasma membrane. Our preliminary data revealed that the exocyst directly interacts with the ESCRT and inhibition of the exocyst in cells block exosome secretion. In this proposal, we will first study the molecular mechanisms by which the biogenesis and release of exosomes is coordinated by different members of the exocyst complex. Second, we will investigate how the secretion of exosomes is regulated during tumor cell invasion; a molecular pathway by which oncogenic Akt promotes exosome secretion through the activation of Rab GTPase will be delineated. Our study will contribute to the understanding of the basic biology of exosomes, and shed light to the mechanisms of tumor cell invasion.